Patients who take opioids for chronic pain typically develop tolerance to these drugs. Now, researchers led by Taka-aki Koshimizu, Jichi Medical University, Shimotsuke, Japan, and Yukio Takano, Fukuoka University, Japan, identify a cluster of molecules underlying morphine tolerance, and show it is possible to ease this outcome, at least in rodents.

They demonstrate that within the rostral ventromedial medulla (RVM), a hindbrain structure involved in the descending modulation of pain, vasopressin 1b receptors (V1bRs) can partner with mu-opioid receptors (MORs). At the same time, they help MORs attach to beta-arrestin-2, a key component of the intracellular pathway associated with opioid side effects. Once formed, this tripartite complex not only heightens pain but also accelerates tolerance.

The study “adds a very interesting piece to our understanding of opioid tolerance,” said Macdonald Christie, University of Sydney, Australia, who was not involved in the new work. But even more importantly, “what it adds is the potential that we might actually be able to modulate it in humans.”

“This paper is a technical tour de force,” said Howard Gutstein, University of Pittsburgh, US, who also did not take part in the research. “It’s super comprehensive.”

Still, by disrupting the complex in mice, the team could only slow the time course of tolerance rather than completely eliminate it. If this finding holds true in patients, that means that “in an acute setting, an antagonist could be used as a sort of adjuvant, to reduce the amount of opiates required to treat pain,” added Gutstein.

The findings were published online April 30 in Nature Neuroscience.

It takes two (receptors)

In 2009, Takano, along with Kenji Honda, also at Fukuoka University and a co-author on the current study, were studying mice genetically modified to lack V1bRs, to test if these receptors controlled nociception (Honda and Takano, 2009). As it turned out, the knockouts were less sensitive to noxious heat compared to their unaltered littermates. But they were also more sensitive to morphine, which yielded a higher level of pain relief after a single dose.

In the new study, the researchers extended these discoveries by tracking how quickly the knockout mice developed tolerance. Following five daily doses of morphine, wild-type mice responded faster during the tail-flick test, a sign of tolerance. In contrast, the knockouts took 15 days to show a similar reduction in their nociceptive reflexes.

To find out why, the group focused on the RVM, one of several brain regions where they detected V1bR transcripts. “The RVM has been known to regulate pain sensitivity at the spinal level,” said Koshimizu.

In membrane samples of the RVM, they specifically examined intracellular levels of cyclic adenosine monophosphate (cAMP). With chronic morphine, the enzyme adenylyl cyclase undergoes “superactivation,” in turn generating more cAMP. This phenomenon, said Koshimizu, has long served as a marker of morphine tolerance. And, it can be suppressed by Gi/o protein signaling, which is triggered by receptors like MORs.

In samples taken from V1bR knockout mice, DAMGO, a MOR agonist, proved to be better at reducing forskolin-stimulated cAMP, compared to samples from wild-type animals. Also, unlike what was seen in the latter case, DAMGO still had an effect even after nine daily doses of morphine in the knockouts. Thus, V1bRs somehow could change the properties of MORs.

Further results supported this idea. First, a fraction of RVM neurons expressing MORs also expressed V1bRs (14 percent), and vice versa (20 percent). Second, using cultured human embryonic kidney (HEK) cells, the researchers could co-immunoprecipitate both receptors. Lastly, by assessing the physical distance between MORs and V1bRs in HEK cells using bioluminescence resonance energy transfer (BRET) imaging, they found that the two molecules were within 10 nanometers of one another.

Despite this proximity, “there is a possibility that they are not interacting directly,” said Koshimizu. “MORs can dimerize through their transmembrane domains. So if V1bRs and MORs form heterodimers, they might do so using transmembrane domains, but we do not have any experimental data that show this yet,” he added.

A molecular complex

As the researchers knew, however, morphine tolerance in mice depends on beta-arrestin-2, which is recruited by MORs after opioids bind to them (Bohn et al., 2000). Consistently, by knocking down the expression of beta-arrestin-2 in HEK cells, they could tamp down forskolin-stimulated cAMP.

Following up on this, they used BRET imaging to measure the distance between MORs and beta-arrestin-2 in HEK cells, with or without V1bRs. Unexpectedly, V1bR acted as a bridge between the two molecules, thanks to a stretch of leucine amino acids in its C-terminal region.

It was this fully formed complex that seemed to feed into tolerance. Using CRISPR-Cas9, a gene editing technique, the researchers removed the leucine-rich segment of V1bR in mice. In doing so, they could recapitulate the phenotype of the V1bR knockouts, enhancing the analgesia from each of five daily doses of morphine.

“There have been some indications over the years, even from the early 1990s, that vasopressin and opioid systems may interact, and that this may influence morphine tolerance,” Christie said. “But they’ve been fairly weak lines of evidence, until this study came out.”

Slowing tolerance

Together, the new findings suggest that a drug capable of accessing the RVM and ultimately interfering with the molecular complex might counteract opioid tolerance in patients, said Christie. Indeed, when injected into the hindbrain of mice or rats before daily morphine injections, the V1bR selective blocker SSR149415 also slowed the development of tolerance. In the past, clinical trials have only evaluated SSR149415 in people with depression.

“It’s important which type of vasopressin receptor is targeted, and they’re clearly saying that in rodents the V1bR subtype is the one that’s important,” said Christie. The V1aR subtype, on the other hand, is not, as tolerance levels of V1aR knockouts given morphine across 15 days matched those of wild-type animals.

Koshimizu, Christie, and Gutstein all envision a drug that would be administered alongside morphine or possibly other opioids, thereby producing a dose-sparing effect—that is, analgesia with a lower than normal dose. “Any dose-sparing effect potentially enhances drug safety,” said Christie.

While a V1bR antagonist might eventually serve as that kind of drug, there are other strong contenders. For example, last year, a study conducted in mice revealed that the peripherally restricted and clinically approved MOR antagonist methylnaltrexone bromide could prevent morphine tolerance (see PRF related story).

For now, Christie remains cautious about predicting whether the approach outlined by Koshimizu and his team will work in humans. “We might not find the mu-opioid receptor and the V1b receptor together in the same relevant neurons in the human nervous system,” he said. Or, “it might turn out to be a limited mechanism, so that it doesn’t broadly apply to a large range of pain patients or all aspects of tolerance. We have to see if this actually pans out.”

Matthew Soleiman is a science writer residing in Nashville, Tennessee. Follow him on Twitter @MatthewSoleiman.